This invention pertains to a method to selectively remove molecules bound to a surface according to their binding strength by attaching micrometer-scale particles to the bound molecules and then applying controlled, laminar fluidic forces to the particles. Such “fluidic force discrimination” (FFD) can be used to improve the sensitivity and selectivity of biochemical binding assays in many fields of use, including forensics, agriculture, medical diagnostics, food and water safety, and anti-terrorism.
Binding assays such as immunoassays, DNA hybridization assays, and receptor-based assays are widely used to detect trace quantities of specific target molecules contained in a complex sample. Typically, a solid substrate is coated with receptor molecules that have a specific binding affinity for the target. When a liquid sample containing the target is applied to the substrate, the target biomolecules are captured onto the surface by molecular recognition. This capture can be accomplished via any specific ligand-receptor combination such as antibody-antigen or other specific binding combination such as complementary sequences of polynucleic acids (DNA, RNA, or PNA).
A common approach to detecting captured target molecules is to chemically attach to them a label that generates an observable signal. For example, a label can include a radioactive isotope, enzyme, fluorescent molecule(s) or magnetic particle. The attachment can be made via any chemical means, but is usually made with very strong attachment chemistry such as covalent or aminated electrostatic bonding, or via high-affinity molecular recognition to a second, exposed part of the captured target molecule. The label is detected by appropriate means as a measure of the concentration of the target in the sample. Detection methods have been developed based on a range of transduction mechanisms, including optical, electrical, magnetic, radioactive, electrochemical, thermal, and piezoelectrical. One example is illustrated in FIG. 1 for a case where a plurality of capture molecules specific to different targets are separately immobilized on a substrate with built-in sensors for the label particles. Note that for micrometer-scale labels this illustration is not to scale: the target and receptor molecules are typically 10 to 1000 times smaller than the labels.
There are many variants to binding assays using labels, but a common goal is to measure the concentration of the target with as much sensitivity and selectivity as possible. As long as a sufficient number of labels are present to generate a detectable signal, the sensitivity and selectivity of a binding assay can be limited by labels bound to the surface but not bound to a captured target (sometime referred to as “background” signal). Such labels may be attached to molecules that were also present in the sample and have bound to the surface through relatively weak, non-specific bonds. Alternately, labels may be directly bound to the surface by buoyant weight and/or non-specific bonds. The ability to selectively remove labels bound non-specifically to the surface can greatly improve the sensitivity and selectivity of a binding assay by lowering the minimum number of labels that can be associated with confidence with the intended target, and reducing the likelihood of wrongly associating detected labels with captured target, respectively.
It is possible to selectively remove labels bound non-specifically to a surface in a binding assay by applying a force to the labels sufficient to break weak, non-specific bonds but too small to remove those labels bound by the stronger bonds of specific molecular recognition. On surfaces specially prepared to inhibit non-specific binding, forces on the order of 1 pN are required for this purpose. It is also possible to use the application of forces to label particles for the purpose of selectively breaking specific bonds of increasing strength and thereby either measure the rupture force or identify the bound target based on the rupture force. Forces >10 pN and as large as 1 nN may be required for this purpose. U.S. Pat. Nos. 5,981,297 and 6,180,418 describe the use of magnetically active beads and magnetic forces to selectively remove non-specifically bound beads (BARC). U.S. Pat. Nos. 6,086,821 and 6,368,553 describe the use of ultrasonic energy to provide a variable force for measuring the binding forces between molecular entities and for sensing the presence of an analyte in a test sample. The above referenced patents are incorporated by reference in their entirety.
In many binding assays it is common practice to use some type of rinsing step for the purpose of reducing the background signal. Common rinsing methods include soaking with our without mechanical agitation and spraying with non-steady flow. Under these poorly controlled conditions smaller particles are removed with greater difficulty because of the no-slip boundary condition at the walls. For sufficiently large fluid boundary dimensions and flow velocities, fluid inertia leads to turbulent flow that can enhance particle removal. At small enough fluid boundary dimensions and velocities, however, the flow is determined by viscosity alone, leading to a steady, laminar flow that does not easily remove particles from surfaces. The two regimes are characterized by the Reynolds number, Re=ρdv/η, where ρ is the fluid density, d is the characteristic dimensions of a channel (volume/surface area), v is the fluid velocity, and η is the viscosity. For particles or channels less than 1 mm wide and velocities less than 1 mm/s, Re is less than 1 and the flow is definitely laminar. The dividing line between laminar and the beginning of turbulent flow is at Re˜2000. Laminar flow conditions commonly occur in blood vessels and in the microfluidics systems used in many biosensor systems.
Viscous hydrodynamic forces on particles at a wall have been studied with the goal of understanding cell adhesion. For example, the migration of white blood cells occurs (via molecular control) as a slow sticky-rolling detachment along blood vessel walls. Chang and Hammer, Langmuir, 1996, 12, 2271-2282 developed a lever amplification model and simulated forces on molecules binding particles to a wall in fluid flow tangential to the binding surface. Zocchi observed the normal force components on 4.5 μm-diameter particles in a tangential flow (Biophysical J. 2001, 81, 2946-2953). He measured a lever amplification of the flow forces and observed the rupture of biotin-streptavidin bonds and applied the results to the interpretation of cell adhesion assays.